Glioma is the most common and most lethal brain tumor, highly resistant to radiation and chemotherapy. To develop more effective treatments, it is vital to better understand the molecular mechanisms of this cancer. We previously reported in gliomas an oncogenic role for Notch, a critical pathway in stem cell maintenance and cell fate determination. In subsequent work now submitted, we showed that other key pathways in gliomas are inhibited by a putative tumor suppressor, microRNA-7. microRNAs are small non-coding RNAs recently found to down-regulate a large subset of human genes, and evidence suggests that some microRNAs have powerful oncogenic or tumor suppressor functions. Our investigation of both the Notch pathway and microRNA led us to assess whether there might be microRNAs opposing the Notch pathway with significant roles in gliomas. Preliminary studies revealed two promising candidates, microRNA-7 and microRNA-326. Both have several predicted targets in the Notch pathway and suppress Notch activity, show decreased expression in human gliomas relative to normal brain, and inhibit viability and invasiveness following transfection into glioma cells. In this application, we propose the systematic investigation of microRNAs -7 and -326 as Notch-inhibiting tumor suppressors with therapeutic potential for glioma. In Aim #1, we will use immunoblotting and 3'-UTR luciferase reporters to validate which of the predicted Notch pathway members are indeed directly targeted by these microRNAs. Additionally, we will determine whether inhibitors of microRNAs-7 and -326 can increase Notch activity and expression of Notch pathway targets. To further investigate down-regulation of these microRNAs in Aim #2, expression of the various forms of these microRNAs will be quantified in human glioma and normal brain samples and their levels correlated to Notch activity and to levels of targeted Notch pathway proteins. We will also perform global microRNA profiling in human gliomas and normal brain to confirm that down-regulation of these microRNAs is selective. In Aim #3, the phenotypic effects of transfecting microRNA- 7 and microRNA-326 into established glioma lines will be evaluated and the contribution of Notch inhibition to these effects determined. Recent evidence has suggested that standard cancer cell lines may be poorly representative of the original tumors, however, and that gliomas and other cancers may be more accurately modeled by lines grown from a small stem cell-like subpopulation within the tumors. It is hypothesized that this stem cell-like fraction is responsible for tumorigenesis and regrowth of tumors, and they are highly resistant to standard therapies. We will therefore determine in Aim #4 the effects of microRNA-7 and microRNA-326 on growth, differentiation, and invasiveness in glioma tumor stem cell lines in vitro as well as in orthotopic xenograft mouse models. Successful completion of the proposed experiments will establish microRNA-7 and microRNA-326 as novel endogenous inhibitors of the Notch pathway with tumor suppressor roles in gliomas, with importance for neuro-oncology and likely relevance for development and for other cancers.